CN105685015B - A kind of cells frozen storing liquid - Google Patents
A kind of cells frozen storing liquid Download PDFInfo
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- CN105685015B CN105685015B CN201610137530.XA CN201610137530A CN105685015B CN 105685015 B CN105685015 B CN 105685015B CN 201610137530 A CN201610137530 A CN 201610137530A CN 105685015 B CN105685015 B CN 105685015B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The present invention provides a kind of cells frozen storing liquid, the component including following volumes score：9~13% dimethyl sulfoxide, 2~7% human serum albumins and 82~87% culture medium；The culture medium includes Lonza basal medium and/or DMEN high glucose medium.Culture medium and human seralbumin egg in the present invention together constitute ingredient and enrich polynary nutrition system, provide stable, direct nutrition supply for cell, ensure that cell survival rate.In addition, the macromolecular substances such as protein form hydration shell during temperature decline, ice crystal is reduced to be formed, cell is avoided because when the temperature drops, cell mechanical damage, death caused by the formation of ice crystal, to improve the Cell viability after through a long time cryopreservation resuscitation.The experimental results showed that the use of cell recovery rate of the frozen stock solution in the present invention after freezing 1 month, 6 months and 12 months being respectively 94~98%, 94~97% and 92~94%.
Description
Technical field
The invention belongs to field of biomedicine technology more particularly to a kind of cells frozen storing liquids.
Background technique
In the passage of cell culture and daily maintenance process, in terms of culture utensil, culture solution and various preparations all
It need to largely expend, and cell once leaves living body and starts originally culture, its various biological natures all will gradually become
Change and constantly has new variation with the increase of passage number and the variation of vitro condition.Therefore cell jelly is carried out in time
It deposits very necessary.
Currently, the most common technology of cell cryopreservation is liquid nitrogen frozen preservation method, it is mainly appropriate protectant using addition
Slow freezing carrys out freeze-stored cell, such as uses glycerol or dimethyl sulfoxide (DMSO) for protective agent, because if cell is being not added
The moisture of directly freezed in any protectant situation, intraor extracellular can quickly form ice crystal, so as to cause a series of bad anti-
It answers, such as：Cell dehydration makes partial electrolysis matter concentration increase, pH value is caused to change, and partially protein is denaturalized for these reasons,
So as to cause cell interior space structure disorder, thus lysosome membrane is damaged and releases lysosomal enzyme, makes to tie into the cell
It constitutes to divide and damage, mitochondrial swelling, function is lost, and causes energy metabolism impairment.Class lipoprotein complex on after birth
Also it is easily destroyed the change for causing permeability of cell membrane, loses cellular content.If intracellular ice crystal formed it is more, with cold
Freeze the reduction of temperature, ice crystal volume expansion causes nucleus DNA steric configuration that irreversible damage occurs, and causes cell death.
The existing scheme that freezes goes to save cell frequently with the scheme that freezes of 10%DMSO+90%FBS, and the program can be very
Good is that zooblast provides nutriment, the cell short time is frozen recover again after remain to maintain higher motility rate.But it should
It is lower that cell cryopreservation scheme through a long time freezes rear cell recovery rate, and only 85% or so, directly restrict the clinic of freeze-stored cell
Using.
Summary of the invention
The purpose of the present invention is to provide a kind of cells frozen storing liquids, are frozen for a long time using cells frozen storing liquid provided by the invention
Cell recovery rate after depositing is higher.
The present invention provides a kind of cells frozen storing liquid, the component including following volumes score：
9~13% dimethyl sulfoxide, 2~7% human serum albumins and 82~87% culture medium；
The culture medium includes Lonza basal medium and/or DMEN high glucose medium.
Preferably, the volume fraction of the dimethyl sulfoxide is 10~12%.
Preferably, the volume fraction of the human serum albumins is 3~6%.
Preferably, the volume fraction of the human serum albumins is 4~5%.
Preferably, the volume fraction of the culture medium is 83~86%.
Preferably, the volume fraction of the culture medium is 84~85%.
The present invention provides a kind of cells frozen storing liquid, the component including following volumes score：9~13% dimethyl is sub-
Sulfone, 2~7% human serum albumins and 82~87% culture medium；The culture medium include Lonza basal medium and/or
DMEN high glucose medium.Culture medium and human seralbumin egg in the present invention together constitute ingredient and enrich polynary nutrition system,
Stable, direct nutrition supply is provided for cell, ensure that cell survival rate.In addition, during temperature decline, egg
The macromolecular substances such as white matter form hydration shell, reduce ice crystal and are formed, avoid cell because when the temperature drops, the formation of ice crystal is made
At cell mechanical damage, death, to improve the Cell viability after through a long time cryopreservation resuscitation.The experimental results showed that making
With cell recovery rate of the frozen stock solution in the present invention after freezing 1 month, 6 months and 12 months be respectively 94~98%, 94~
97% and 92~94%.
Specific embodiment
The present invention provides a kind of cells frozen storing liquid, the component including following volumes score：
9~13% dimethyl sulfoxide, 2~7% human serum albumins and 82~87% culture medium；
The culture medium includes Lonza basal medium and/or DMEN high glucose medium.
Cell recovery rate after being frozen for a long time using cells frozen storing liquid provided by the invention is higher.
In the present invention, the volume fraction of the dimethyl sulfoxide (DMSO) is 9~13%, preferably 10~12%, more
Preferably 10%.The present invention does not have special limitation to the source of the dimethyl sulfoxide, using conventional commercial goods.
In the present invention, the volume fraction of the human serum albumins (HSA) is 2~7%, preferably 3~6%, more excellent
It is selected as 4~5%；The present invention does not have special limitation to the source of the human serum albumins, specifically, in implementation of the invention
In example, the human serum albumins that the volume fraction of Guangzhou pharmaceuticals offer is 20% can be used.
In the present invention, the volume fraction of the culture medium be 82~87%, preferably 83~86%, more preferably 84~
85%；The culture medium includes Lonza basal medium and/or DMEN high glucose medium, and the present invention trains the basis Lonza
The source for supporting base does not have special limitation, specifically, in an embodiment of the present invention, it is limited that the high match biotechnology in Guangzhou can be used
The Lonza basal medium that company provides.The present invention does not have special limitation to the source of the DMEN high glucose medium, specifically
, the DMEN high glucose medium that the specification of Gibco company offer is 500mL/ bottles can be used.
Dimethyl sulfoxide and mass concentration is added in the medium as 20% human serum albumins, is finally contained body
The dimethyl sulfoxide of fraction 9~13%, 2~7% human serum albumins and 82~87% culture medium cells frozen storing liquid.
In the present invention, the source of the culture medium, dimethyl sulfoxide and dextran and dosage and above-mentioned technical proposal
Middle culture medium, dimethyl sulfoxide and the source of dextran are consistent with dosage, and details are not described herein.
Cells frozen storing liquid provided by the invention can be used for freezing a plurality of types of human body cells, such as human hepatocyte, human body
Immunocyte, human body tumour cell or human body adult cell, specifically, in an embodiment of the present invention, can be used with Types Below
With the human body cell in source：
According to document：Tian Lin, Sun Xiaofang, Haibo Liu human adipose-derived stem cell are separately cultured and biological property《China
Tissue Engineering Study》, 2012,16 (32)：Method in 5946-5952 obtains ADSCs primary cell, by secondary culture, obtains
The cell of the P3~P5 taken；
According to document：The improvement of mono-, people's Mesenchymal Stem Cells from Umbilical Cord isolated culture method of Li Yanqi, Wang Hong《Chinese group
Knit engineering research》, 2014,18 (10):Method in method in 1609-1614 obtains UC-MSC primary cell, by passage
Culture, the cell of P3~P5 of acquisition.
According to document：Chen Dan, the separation of tri- kinds of the separation human peripheral blood mononuclear cell's methods in king small east《Medical University Of Tianjin
Journal》, 2014.6:Method in 483-485 obtains human peripheral blood single nucleus cell (PBMC)；
Tumour cell K562, HL60 cell are obtained from Ji'nan University's school of life and health sciences；
According to document：In-vitro separation, purifying culture and the cell mirror of Wang Lingling, Ma Feng, Zhang Yucheng human fibroblasts
Determine《Aged in China magazine》, 2012,32:Method in 1215-1216. obtains fibroblast, after secondary culture,
Obtain the cell of P3~P5.
The present invention is respectively by after above-mentioned cell cryopreservation, in 1 month, 6 months and 12 months by cell recovery, the results showed that,
The Cell viability recovered after freezing 12 months is 93% or more.In the present invention, the cell cryopreservation and the method for recovery are
The common method of those skilled in the art.
The present invention provides a kind of cells frozen storing liquid, the component including following volumes score：9~13% dimethyl is sub-
Sulfone, 2~7% human serum albumins and 82~87% culture medium；The culture medium include Lonza basal medium and/or
DMEN high glucose medium.Cells frozen storing liquid provided by the invention has the following advantages that：
The present invention avoids cell from causing in frozen storage process because of ice crystal formation as far as possible by optimization cell cryopreservation scheme
Cell mortality；
Optimize the system of freezing, cell after recovery can direct feedback to human body；
The serum for not adding animal origin avoids the virus pollution of animal origin；
Frozen stock solution in the present invention can be used for various kinds of cell type, and such as stem cell, immunocyte, tumour cell, adult is thin
Born of the same parents etc..
In order to further illustrate the present invention, detailed to a kind of cells frozen storing liquid progress provided by the invention with reference to embodiments
Thin description, but limiting the scope of the present invention cannot be understood as.
In the examples below, Lonza basal medium is provided by Guangzhou Ang Sai Bioisystech Co., Ltd, Lonza base
Basal culture medium is 500mL/ bottles, and human serum albumins is provided by Guangzhou pharmaceuticals, mass concentration 20%.
In the examples below, involved percentage is percentage by volume.
1 human stem cell ADSCs's of embodiment freezes
The human serum albumins of DMSO and mass concentration 20% are separately added into Lonza basal medium, formation contains
The cells frozen storing liquid of 10%DMSO and 5% human serum albumins.
According to document：Tian Lin, Sun Xiaofang, Haibo Liu human adipose-derived stem cell are separately cultured and biological property《China
Tissue Engineering Study》, 2012,16 (32)：Method in 5946-5952 obtains ADSCS primary cell and obtains by secondary culture
The cell of the P3~P5 taken.
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.Cell is resuspended with 4 DEG C of cells frozen storing liquids, takes 50 μ L
Cell suspension, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing and quantity calculates；Adjustment
Cell density is 1 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube, 1.5mL/ pipe；It is marked
Note, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, the cell of 12 months recovery ADSCs, two pipe of recovery every time；Specific step is as follows：From
Cell is taken out in liquid nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively and is trained containing the basis 10mL Lonza
In the 50mL centrifuge tube for supporting base, 200g is centrifuged 5min, is resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months ADSCs Cell viabilities (being repeated 3 times counting), and the results are shown in Table 1, table 1
Cell viability after the cell recovery frozen for the embodiment of the present invention 1~6.
The present invention is taken at 1 month, 6 months, 12 months multiple Soviet Union's ADSCs cells respectively, is resuspended with 10%FBS+PBS, and
It is adjusted to 1 × 106The cell suspension of cells/mL takes the monoclonal antibody of anti-human CD73, CD105, CD45 and HLA-DR respectively
200 μ L of cell suspension is added in each 5 μ L, is protected from light is incubated for 20min at room temperature, while setting up Isotype control group, 1500r/min centrifugation
5min abandons supernatant, is washed 2 times with the PBS containing 10%FBS, upper machine testing recovery after being resuspended with 500 μ L, 1640 basal medium
Cell surface antigen afterwards.The result shows that cell surface antigen expression complies with standard.
2 human stem cell UC-MSC's of embodiment freezes
According to document：The improvement of mono-, people's Mesenchymal Stem Cells from Umbilical Cord isolated culture method of Li Yanqi, Wang Hong《Chinese group
Knit engineering research》, 2014,18 (10):Method in method in 1609-1614 obtains UC-MSC primary cell, by passage
Culture, the cell of P3~P5 of acquisition.
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.With cells frozen storing liquid obtained in 4 DEG C of embodiment 1
Cell is resuspended, the cell suspension of 50uL is taken, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing
And quantity calculates；Adjustment cell density is 1 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube,
1.5mL/ pipe；It is marked, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, the cell of 12 months recovery UC-MSC, two pipe of recovery every time；Specific step is as follows：From
Cell is taken out in liquid nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively and is cultivated containing the basis 10mLLonza
In the 50mL centrifuge tube of base, 200g is centrifuged 5min, is resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months UC-MSC Cell viabilities (being repeated 3 times counting), and the results are shown in Table 1, table
Cell viability after 1 cell recovery frozen for the embodiment of the present invention 1~6.
The present invention is taken at 1 month, 6 months, 12 months UC-MSC cells recovered respectively, is resuspended with 10%FBS+PBS, and
It is adjusted to 1 × 106The cell suspension of cells/mL takes the monoclonal antibody of anti-human CD73, CD105, CD45 and HLA-DR respectively
200 μ L of cell suspension is added in each 5 μ L, is protected from light is incubated for 20min at room temperature, while setting up Isotype control group, 1500r/min centrifugation
5min abandons supernatant, is washed 2 times with the PBS containing 10%FBS, upper machine testing recovery after being resuspended with 500 μ L, 1640 basal medium
Cell surface antigen afterwards.The result shows that cell surface antigen expression complies with standard.
Embodiment 3 human peripheral blood single nucleus cell (PBMC's) freezes
According to document：Chen Dan, the separation of tri- kinds of the separation human peripheral blood mononuclear cell's methods in king small east《Medical University Of Tianjin
Journal》, 2014.6:Method in 483-485 obtains human peripheral blood single nucleus cell (PBMC)；
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.With cells frozen storing liquid obtained in 4 DEG C of embodiment 1
Cell is resuspended, the cell suspension of 50uL is taken, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing
And quantity calculates；Adjustment cell density is 3 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube,
1.5mL/ pipe；It is marked, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, the cell of 12 months recovery PBMC, two pipe of recovery every time；Specific step is as follows：From liquid
Cell is taken out in nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively and is cultivated containing the basis 10mL Lonza
In the 50mL centrifuge tube of base, 200g is centrifuged 5min, is resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months PBMC Cell viabilities (being repeated 3 times counting), and the results are shown in Table 1, table 1
Cell viability after the cell recovery frozen for the embodiment of the present invention 1~6.
The present invention is resuspended in 1 month, 6 months, the PBMC of recovery in 12 months with 10%FBS+PBS, and it is adjusted to 1 ×
106The cell suspension of cells/mL takes each 5 μ L of the monoclonal antibody of anti-human CD3, CD56, CD4, CD8, CD19 respectively, is added thin
200 μ L of born of the same parents' suspension is protected from light is incubated for 20min at room temperature, while setting up Isotype control group, and 1500r/min is centrifuged 5min, abandons supernatant,
It is washed 2 times with the PBS containing 10%FBS, cell surface is anti-after upper machine testing recovery after being resuspended with 500 μ L, 1640 basal medium
It is former.The result shows that cell surface antigen expression complies with standard.
4 human body tumour cell K562 cell of embodiment freezes
Tumour cell K562 cell is obtained from Ji'nan University's school of life and health sciences；
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.With cells frozen storing liquid obtained in 4 DEG C of embodiment 1
Cell is resuspended, the cell suspension of 50 μ L is taken, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing
And quantity calculates；Adjustment cell density is 5 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube,
1.5mL/ pipe；It is marked, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, the cell of 12 months recovery K562, two pipe of recovery every time；Specific step is as follows：From liquid
Cell is taken out in nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively containing 10mLLonza basal medium
50mL centrifuge tube in, 200g is centrifuged 5min, be resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months K562 Cell viabilities (being repeated 3 times counting), and the results are shown in Table 1, table 1
Cell viability after the cell recovery frozen for the embodiment of the present invention 1~6.
The present invention is resuspended in 1 month, 6 months, the K562 cell of recovery in 12 months with 10%FBS+PBS, and it is adjusted to 1 ×
106The cell suspension of cells/mL takes each 5 μ L of the monoclonal antibody of anti-human CD13, CD117, CD34 and HLA-DR respectively, is added
200 μ L of cell suspension is protected from light is incubated for 20min at room temperature, while setting up Isotype control group, and 1500r/min is centrifuged 5min, in abandoning
Clearly, it is washed 2 times with the PBS containing 10%FBS, cell surface after upper machine testing recovery after being resuspended with 500 μ L, 1640 basal medium
Antigen.The result shows that cell surface antigen expression complies with standard.
5 human body tumour cell HL60 cell of embodiment freezes
Tumour cell HL60 cell is obtained from Ji'nan University's school of life and health sciences；
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.With cells frozen storing liquid obtained in 4 DEG C of embodiment 1
Cell is resuspended, the cell suspension of 50uL is taken, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing
And quantity calculates；Adjustment cell density is 5 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube,
1.5mL/ pipe；It is marked, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, the cell of 12 months recovery HL60, two pipe of recovery every time；Specific step is as follows：From liquid
Cell is taken out in nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively containing 10mLLonza basal medium
50mL centrifuge tube in, 200g is centrifuged 5min, be resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months HL60 Cell viabilities (being repeated 3 times counting), and the results are shown in Table 1, table 1
Cell viability after the cell recovery frozen for the embodiment of the present invention 1~6.
The present invention is resuspended in 1 month, 6 months, the HL60 cell of recovery in 12 months with 10%FBS+PBS, and it is adjusted to 1 ×
106The cell suspension of cells/mL takes each 5 μ L of the monoclonal antibody of anti-human CD13, CD11b respectively, and 200 μ of cell suspension is added
L is protected from light is incubated for 20min at room temperature, while setting up Isotype control group, and 1500r/min is centrifuged 5min, abandons supernatant, with containing 10%FBS
PBS wash 2 times, cell surface antigen after upper machine testing recovery after being resuspended with 500 μ L, 1640 basal medium.The result shows that
Cell surface antigen expression complies with standard.
6 human fibroblasts of embodiment freeze
According to document king's tinkling of pieces of jades, Ma Feng, in-vitro separation, purifying culture and the cellular identification of Zhang Yucheng human fibroblasts
《Aged in China magazine》, 2012,32:Method in 1215-1216. obtains fibroblast, after secondary culture, obtains
The cell of P3~P5；
1500r/min is centrifuged 5min after being resuspended with PBS, abandons supernatant.With cells frozen storing liquid obtained in 4 DEG C of embodiment 1
Cell is resuspended, the cell suspension of 50uL is taken, by cell steep cliff (v):0.4% trypan blue (v)=1:Cell viability is carried out after 1 mixing
And quantity calculates；Adjustment cell density is 3 × 10 to density is frozen6Cells/mL dispenses cell suspension into cryopreservation tube,
1.5mL/ pipe；It is marked, freezes 6 pipes.
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid
Nitrogen.
Respectively at 1 month, 6 months, 12 months recovery fibroblasts, recovery two was managed every time；Specific step is as follows：From liquid
Cell is taken out in nitrogen, is dissolved rapidly in 37 DEG C of water, and cell suspension is transferred to respectively and is cultivated containing the basis 10mL Lonza
In the 50mL centrifuge tube of base, 200g is centrifuged 5min, is resuspended after abandoning supernatant with lonza basal medium, and adjust density to 1 ×
106cells/mL。
The present invention calculates separately 1,6 and 12 months fibroblast motility rates (being repeated 3 times counting), and the results are shown in Table 1, table
Cell viability after 1 cell recovery frozen for the embodiment of the present invention 1~6.
Present invention 10%FBS+PBS be resuspended 1 month, 6 months, the fibroblasts of recovery in 12 months and be adjusted to 1 ×
106The cell suspension of cells/mL takes anti-human vimentin antibodies (Vimentin) and keratin antibody (CK15 antibody) respectively
Each 5 μ L of monoclonal antibody, 200 μ L of cell suspension is added, is protected from light is incubated for 20min at room temperature, while setting up Isotype control group,
1500r/min is centrifuged 5min, abandons supernatant, is washed 2 times with the PBS containing 10%FBS, after being resuspended with 500 μ L, 1640 basal medium
Cell surface antigen after upper machine testing recovery.The result shows that cell surface antigen expression complies with standard.
Cell viability after the cell recovery that 1 embodiment of the present invention 1~6 of table freezes
As can be seen from Table 1, different types of human body cell is frozen using the cells frozen storing liquid in the present invention, even across
It freezes for a long time, Cell viability still with higher after recovery.93% or more, highest can reach 94% or so.
Embodiment 7~12
The human serum albumins of DMSO and mass concentration 20% are separately added into Lonza basal medium, formation contains
The cells frozen storing liquid of 10%DMSO and 3% human serum albumins.
Using above-mentioned cells frozen storing liquid, human stem cell ADSCs is frozen respectively according to the cryopreservation methods in Examples 1 to 6
(embodiment 7), human stem cell UC-MSC (embodiment 8), human peripheral blood single nucleus cell (PBMC) (embodiment 9), human body are swollen
Oncocyte K562 cell (embodiment 10), human body tumour cell HL60 cell (embodiment 11) and human fibroblasts (embodiment
12), and the Cell viability (being repeated 3 times counting) of each cell in embodiment 7~12 has been calculated separately, the results are shown in Table 2, table 2
Cell viability after the cell recovery frozen for the embodiment of the present invention 7~12.
Cell viability after the cell recovery that 2 embodiment of the present invention 7~12 of table freezes
| Embodiment | Cell type | Freeze preceding Cell viability | 1 month Cell viability | 6 months Cell viabilities | 12 months Cell viabilities |
| 7 | ADSCs | 98.32±2.54 | 96.37±6.35 | 93.46±7.25 | 88.28±8.09 |
| 8 | UC-MSC | 99.46±1.87 | 96.73±7.02 | 93.51±7.36 | 87.67±6.48 |
| 9 | PBMC | 96.55±3.60 | 95.91±8.64 | 92.48±8.53 | 86.51±8.25 |
| 10 | K562 | 99.23±1.93 | 97.14±6.47 | 94.29±8.28 | 88.83±8.08 |
| 11 | HL60 | 97.97±2.69 | 96.22±7.58 | 93.26±9.47 | 87.64±8.72 |
| 12 | Fibroblast | 96.42±3.06 | 96.43±8.19 | 93.38±9.15 | 85.49±9.38 |
Embodiment 13~18
The human serum albumins of DMSO and mass concentration 20% are separately added into Lonza basal medium, formation contains
The cells frozen storing liquid of 12%DMSO and 6% human serum albumins.
Using above-mentioned cells frozen storing liquid, human stem cell ADSCs is frozen respectively according to the cryopreservation methods in Examples 1 to 6
(embodiment 13), human stem cell UC-MSC (embodiment 14), human peripheral blood single nucleus cell (PBMC) (embodiment 15), human body
Tumour cell K562 cell (embodiment 16), human body tumour cell HL60 cell (embodiment 17) and human fibroblasts (are implemented
Example 18), and the Cell viability (being repeated 3 times counting) of each cell in embodiment 13~18 has been calculated separately, the results are shown in Table 3,
Table 3 is the Cell viability after the cell recovery that the embodiment of the present invention 13~18 freezes.
Cell viability after the cell recovery that 3 embodiment of the present invention 13~18 of table freezes
| Embodiment | Cell type | Freeze preceding Cell viability | 1 month Cell viability | 6 months Cell viabilities | 12 months Cell viabilities |
| 13 | ADSCs | 98.32±2.54 | 92.65±6.42 | 89.49±8.15 | 83.82±8.31 |
| 14 | UC-MSC | 99.46±1.87 | 92.73±6.79 | 87.66±7.84 | 81.71±7.76 |
| 15 | PBMC | 96.55±3.60 | 90.94±6.49 | 86.68±6.97 | 83.29±8.25 |
| 16 | K562 | 99.23±1.93 | 93.88±7.40 | 88.47±7.83 | 82.59±8.46 |
| 17 | HL60 | 97.97±2.69 | 91.19±7.65 | 85.24±7.48 | 79.07±7.89 |
| 18 | Fibroblast | 96.42±3.06 | 90.08±7.98 | 86.58±8.41 | 81.35±8.63 |
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (1)
1. a kind of cells frozen storing liquid is freezing the application in human body fibroblast, the specific steps are：
The human serum albumins of DMSO and mass concentration 20% are separately added into Lonza basal medium, formation contains 10%
The cells frozen storing liquid of DMSO and 5% human serum albumins；
1500r/min is centrifuged 5min after the human body fibroblast of P3~P5 is resuspended with PBS, abandons supernatant；With 4 DEG C of above-mentioned system
Cell is resuspended in the cells frozen storing liquid obtained, the cell suspension of 50 μ L is taken, by cell suspension and 0.4% trypan blue volume ratio=1:1 is mixed
Cell viability is carried out after even and quantity calculates；Adjustment cell density is 3 × 10 to density is frozen6Cells/mL, cell suspension
Packing is into cryopreservation tube, 1.5mL/ pipe；It is marked, freezes 6 pipes；
Cryopreservation tube equipped with cell suspension is put into freezing storing box, is put into -80 DEG C of refrigerators, after 48h, is transferred to liquid nitrogen.
Priority Applications (1)
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| CN106047702B (en) * | 2016-08-15 | 2018-07-20 | 北京昱龙盛世生物科技有限公司 | A kind of kit and cultural method for cultivating fat stem cell |
| CN107771780A (en) * | 2016-08-29 | 2018-03-09 | 灏灵赛奥（天津）生物科技有限公司 | CAR-T cell cryopreservation medium and cryopreservation method |
| CN108235981B (en) * | 2016-12-23 | 2021-07-23 | 西比曼生物科技（香港）有限公司 | Cell cryopreservation liquid capable of being used clinically |
| CN109221092A (en) * | 2018-11-14 | 2019-01-18 | 广东华夏健康生命科学有限公司 | A kind of cells frozen storing liquid and its application |
| CN109362708A (en) * | 2018-11-15 | 2019-02-22 | 广州南医大生物工程有限公司 | A kind of stem cell refrigerant and its application |
| CN110583622A (en) * | 2019-08-30 | 2019-12-20 | 依科赛生物科技（太仓）有限公司 | T cell serum-free freezing medium and use method thereof |
| CN112335648A (en) * | 2020-11-12 | 2021-02-09 | 安徽惠恩生物科技股份有限公司 | Cell cryopreservation liquid |
| CN113383768B (en) * | 2021-05-22 | 2022-07-05 | 武汉仝干医疗科技股份有限公司 | Hepatocyte freezing solution and preparation method and application thereof |
| CN113455496A (en) * | 2021-06-30 | 2021-10-01 | 苏州大学 | Egg white cell cryopreservation liquid, preparation method and application thereof |
| CN114190364B (en) * | 2021-12-28 | 2023-02-24 | 南京诺唯赞生物科技股份有限公司 | Hybridoma cell cryopreservation liquid and preparation method and application thereof |
| CN114958724A (en) * | 2022-06-26 | 2022-08-30 | 依雨大健康科技(广州)有限公司 | Production method of stable primary hepatocyte kit |
| CN116724992B (en) * | 2023-01-28 | 2024-07-23 | 宁波熙宁检测技术有限公司 | Whole blood frozen stock solution and preparation method and application thereof |
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